JPH01128911A - Skin cosmetic - Google Patents

Abstract

PURPOSE:To obtain a cosmetic having excellent stability with day and adpatability, comprising modified superoxide dismutase prepared by bonding a polysaccharide to superoxide dismutase of copper or zinc type and a water-soluble polyhydric alcohol as essential components. CONSTITUTION:A skin cosmetic capable of preventing drying and aging of skin without causing chapped skin, comprising modified superoxide dismutase (preferably 0.00001-1pt.wt. calculated as enzyme amount) prepared by bonding a polysaccharide such as dextran, starch, chitin or pectin to superoxide dismutase of copper or zinc type and a water-soluble polyhydric alcohol (preferably 0.5-50wt.% based on total amount) as essential components. A polysaccharide having 1,000-100,000mol.wt. is especially preferable as the polysaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a skin cosmetic that can be applied to the face and the like.

[Conventional technology]

Superoxide dismutase is found in animals and plants.

Superoxide dismutase is an enzyme that is widely distributed in living organisms such as microorganisms, and recently several cosmetics have been proposed that utilize the enzymatic action of superoxide dismutase.

[Problem that the invention seeks to solve]

However, if superoxide dismutase is used as it is in cosmetics, the enzyme activity tends to decrease during the production of cosmetics, ■ the color and odor of cosmetics tend to change orally due to the enzyme, and ■ the enzyme This causes problems such as irritation and allergy to the skin, and a solution to these problems has been desired.

The present invention was made in view of the above circumstances, and an object thereof is to provide a modified superoxide dismutase-containing skin cosmetic with improved oral stability and skin compatibility.

[Means for solving the problems] In order to achieve the above object, the skin cosmetic of the present invention has the following features:
Copper-2-zinc type superoxide dismutase is 11! It has a structure in which the essential components are modified superoxide dismutase, which is a combination of the like, and a water-soluble polyhydric alcohol.

[Effect]

In other words, the skin cosmetic of the present invention contains a modified superoxide dismutase in which polyIII is bonded to a copper-zinc type superoxide dismutase, and therefore has improved oral stability and skin compatibility, which have been problems in the past. has been improved.

Next, this invention will be explained in detail.

The copper and zinc type superoxide dismutase used in this invention is a known substance and can be obtained from plant leaves, animal blood cells, liver, placenta, etc. This enzyme is absorbed transdermally and acts on fibroblasts in the dermis and basal cells in the epidermis, exerting excellent anti-aging effects.

Examples of multi-T, V used to modify the superoxide dismutase include dextran, starch, pullulan, inulin, xanthan gum, chitin, mannan,
Examples include cellulose, pectin, carrageenan, alginic acid, etc., and they may be used alone or in combination. The type and molecular weight of these polyjams are not particularly limited, and from the viewpoint of antigenic suppression, those with a molecular ff of 12,000 or more are desirable. Moreover, among these polysaccharides, dextran is particularly suitable because it is highly safe and has excellent oral stability in water. From the viewpoint of effectiveness, its molecular weight is 1,000 to 100,000, especially 60,000 to 9.
0000 is preferred.

The modified superoxide dismutase of the present invention includes the above-mentioned superoxide dismutase and the above-mentioned polyester! For example, it can be produced as follows. That is, first, the polysaccharide is activated by binding with a suitable binding agent. And the above active Hojo tJ! Superoxide dismutase is modified by binding the active site of M to the amino group of superoxide dismutase.

After the above-mentioned modification reaction, the excess active groups of the activated polyalkylene glycol are post-treated by reacting them with a compound containing an amine group, such as lysine, glycine, or aminoethanol.

Further, remaining unreacted binder and the like are separated and removed by ultrafiltration or the like.

In this way, the desired modified superoxide dismutase can be produced.

Compared to the above manufacturing method, more IJ! The binding agent and binding method for activating the enzyme are not particularly limited, and any binding agent may be used as long as the reaction conditions do not deactivate the enzyme. For example, a method in which a polysaccharide activated using a reagent such as cyanogen bromide is reacted with the amino group of an enzyme to form a bond, or a polysaccharide is oxidized with periodic acid to create an aldehyde group and then the amino group of the enzyme is A method of bonding to a cyanurated polysaccharide using cyanuric chloride as a binding agent and then bonding it to an amino group of an enzyme. A method in which a carboxyl group is introduced into a multi-W group via a carboxymethyl group or an aldehyde group, and then a carboxyl group is bonded to the enzyme's amino group using bromoacetylation or glutaraldehyde. Examples include a method of combining them. In addition, the carboxyl group-introduced polysaccharide (the polysaccharide having a carboxyl group is as it is) and the amino group of the enzyme may be bonded using a binding agent such as carbodiimide, or the carboxyl group may be - They may be bonded after further active esterification with hydroxysuccinimide or the like.

The modification reaction between superoxide dismutase and polysaccharide is preferably carried out in a neutral or weakly alkaline solution at room temperature in order to prevent deactivation of the enzyme. In addition, the blending ratio of the superoxide dismutase and polysaccharide varies depending on the molecular weight and structure of the polysaccharide used.
Although not necessarily limited, considering the compatibility with the skin, many tJ3i! It is preferable that ffi is more than 0.5 times the amount of superoxide dismutase on a weight basis.

The modified superoxide dismutase thus obtained maintains high activity and, moreover, has almost or completely suppressed antigenicity.

The water-soluble polyhydric alcohol used in this invention together with the above-mentioned modified superoxide dismutase is one that functions to suppress the oral deterioration of the above-mentioned modified superoxide dismutase in an aqueous solution, such as ethylene glycol and propylene glycol.

Examples include 1.3-butylene glycol, 1.4-butylene glycol, dibutylene glycol, glycerin, diglycerin, glucose, maltol, maltitol, sucrose, fructol, xylitol, sorbitol, maletriose, threitol, and erythritol. These may be used alone or in combination of two or more.

The skin cosmetic of the present invention can be produced in the same manner as ordinary cosmetics using the modified superoxide dismutase and a water-soluble polyhydric alcohol. For example, a skin lotion can be obtained by uniformly mixing modified superoxide dismutase and a water-soluble polyhydric alcohol in purified water. In addition, cosmetic emulsions, cosmetic creams, etc. can be obtained by combining oily substances and emulsifiers. Of course, additives such as colorants, preservatives, ultraviolet absorbers, and antioxidants can be appropriately blended into the above-mentioned various cosmetics as required.

In the above manufacturing method, the amount of modified superoxide dismutase is 0.00001% of the total amount of enzyme.
It is preferable to set the value to 1% (hereinafter abbreviated as "%"). That is, the modified superoxide dismutase is o, o o o.

This is because if it is less than 1%, the enzymes in the resulting skin cosmetic will not function sufficiently, and if it exceeds 1%, the skin cosmetic will tend to be more irritating to the skin.

In addition, the amount of water-soluble polyhydric alcohol blended is 0% of the total amount.
．． It is suitable to set it to be 5 to 50%.

If it is less than 0.5%, the oral stability of the skin cosmetic will be lowered and discoloration and odor will likely occur, while if it exceeds 50%, the skin cosmetic will feel sticky and undesirable.

Further, as the purified water used in the above production method, deionized purified water treated with an ion exchange resin, distilled water, etc., which are usually used in the production of cosmetics, are used.

The blending amount is usually about 40 to 99%.

When the skin cosmetics obtained in this way are used, superoxide, which is generated in large amounts when the skin is exposed to ultraviolet rays or injured, is disproportionated and differentiated by the action of modifying superoxide dismutase, so superoxide It can prevent skin dryness and aging caused by oxides. Furthermore, since superoxide dismutase is modified with polysaccharide, the antigenicity of superoxide dismutase itself is removed, and it does not cause rough skin. Moreover, the modified superoxide dismutase does not deteriorate orally due to the presence of water-soluble polyhydric alcohol. Therefore, the skin cosmetic of the present invention can be used safely for a long period of time and is highly effective.

Next, the present invention will be explained based on examples.

The measurement of enzyme activity, the rough skin improvement effect test, the keratin improvement effect test, the practical test, and the enzymatic activity stability test over time shown in Examples were conducted as follows.

Measurement of activity> Cytochrome C and an enzyme or a modifying enzyme were coexisted in a superoxide production system using xanthine-xanthine oxidase, and the activity of the enzyme was measured using the amount that reduces the reduction rate of cytochrome C as an index.

<Rough skin improvement effect test> The effect of continuous application for 4 weeks was investigated on 20 middle-aged and elderly subjects with rough skin on the lower m. Approximately 1 g of the sample was applied to the test site of the left lower leg of the subject once a day, and the condition of the skin before and after the test was judged according to the following criteria.

No sample was applied to the right lower leg, which served as a control.

*Judgment criteria for skin dryness* -2 Normal ±: Slight dryness, no scaling +: Dryness, mild scaling + 12 Dryness, moderate scaling + 10: Dryness, significant scaling, and test and control areas before and after the test The results were compared, and if the skin dryness improved by two or more levels (for example, +→-1++→±), it was considered valid, if it improved by one level, it was considered somewhat valid, and if there was no change, it was considered invalid. . The test results are shown as the number of subjects who found the drug to be effective or somewhat effective.

<Test on improvement effect on dead skin cells> A mending tape (manufactured by Chiban Co., Ltd.) was adhered to the subject's skin before and after the start of the above-mentioned rough skin improvement effect test, and when the tape was peeled off, the condition of the dead skin cells attached to the tape was observed using a scanning electron microscope. investigated in detail.

Then, the anti-exfoliation property of skin corneocytes was analyzed according to the following criteria, and the corneum-improving effect was determined.

*Judgment criteria for keratin improving effect (increased anti-exfoliation property of corneocytes)* Evaluation points: 1 No scale observed 2: Small scales scattered 3: Small to medium scales noticeable 4: Large scales markedly evaluated after 4 weeks of continuous application If the difference between the evaluation score of the test site and that of the control point was 2 or more points, it was considered valid, if it was 1 point, it was considered somewhat valid, and if it was 0 points, it was considered invalid.

The judgment results were expressed as the number of subjects who found the test to be effective or somewhat effective.

<Practical test> Female test subjects (35-5
The sample was applied to 20 children (5 years old) twice a day, once in the morning and once in the evening, for 3 consecutive months, and the following items were evaluated immediately thereafter.

*Evaluation Items* Skin wettability: Number of people who answered that their skin felt moisturized Stickiness: Number of people who answered that the sample felt sticky Skin smoothness: Number of people who answered that their skin became smooth Elasticity: Number of people who answered that their skin felt sticky Irritation: Number of people who said they felt irritation when applying rash Number of people who said they got a rash between two uses After being left in a constant temperature bath of C for 3 months, the presence or absence of changes in color and odor was observed.

[Example 1] ■ Preparation of modified superoxide dismutase 2.0 g of dextran (average molecular weight 60,000 to 90,000) was
After dissolving in 0 mfl of water, 1N-NaOH was added to adjust the pH to 10.4. and.

To a cyanogen bromide aqueous solution in which 500 μm of cyanogen bromide was dissolved in 6 d of water, the above dextran aqueous solution was gradually added at room temperature of 25°C, and at the same time, 1N-NaOH was added to adjust the pH of the reaction system.
Care was taken to keep the value around 10. After completing this operation in about 15 minutes, it was stirred for about 10 minutes, and then 4% NaH
A CO, aqueous solution was added to adjust the pH to 9. In this way, an activated dextran solution activated with cyanogen bromide was obtained.

On the other hand, copper and zinc type superoxide dismutase obtained from viable blood cells (manufactured by Sigma, 2350 U/mg) 40
0■ was dissolved in 20 d of water, added to the above activated dextran solution, and stirred at room temperature of 25°C overnight. After adding 3.7 g of glycine to the resulting reaction solution and performing a treatment reaction for 2 hours, the solution was purified by ultrafiltration, condensed, and lyophilized to obtain modified superoxide dismutase.

The activity retention rate of this modified enzyme was 80%.

■ Preparation of skin lotion
A skin lotion was obtained by uniformly mixing these raw materials with the following raw material composition using ODJ (abbreviated as ODJ).

Modified SOD 0.01% Glycerin l O, OO Purified water 89.99 [Example 2] ■ Preparation of modified SOD Dextran (average molecular weight 60,000-90,000)
loOmg O, 1M-Na 10. ? After dissolving in 10 ml of water and stirring at room temperature for 16 hours, the resulting reaction solution was purified and concentrated by ultrafiltration. After adding 0.075 M ~ phosphate buffer (pH 7, 8) to make the total volume 5 all, copper, zinc type superoxide dismutase (manufactured by Sigma, 23500/Il) was added.
1g) 20mg dissolved in the same phosphate buffer 5d as above was added and reacted at room temperature for 16 hours.

Next, 5 mol of N a B H40,000 was added to carry out a reduction treatment, and this treated solution was purified and concentrated by ultrafiltration to obtain modified superoxide dismutase.

The activity retention rate of this modified enzyme was 65%.

(2) Preparation of skin lotion Using the above-mentioned modified SOD, a skin lotion was obtained by uniformly mixing the following raw material compositions.

Modified SOD 0.0001% dipropylene glycol 1.0000 Purified water
98.9999 [Example 3] (1) Preparation of modified SOD Dextran with an average molecular weight of 17,900 was used. Modified SOD was obtained in the same manner as in Example 1 except for the above.

The activity retention rate of this modified enzyme was 77%.

■Preparation of skin lotion Using the above modified SOD, make the raw material composition as shown below.
A skin lotion was obtained by uniformly mixing these.

Modified SOD 0.1%1.3-
Butylene glycol 10.0〃Purified water
89.9 [Example 4] ■ Preparation of modified SOD Inulin (average molecular weight 5000) instead of dextran
was used. Other than that, modified SOD in the same manner as in Example 1.
I got it.

The activity retention rate of this modified enzyme was 80%.

(2) Preparation of skin lotion Using the modified SOD described above, a skin lotion was obtained by uniformly mixing the raw materials having the following raw material composition.

Modified SOD 0.1%1.3-
Butylene glycol 5.0〃Purified water
94.9 [Example 5] ■ Preparation of modified SOD Carboxymethyl cellulose 200 ■ 20 tail
of water, and add 1N-HCl! , and pH
4,75, and then 380 ■-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Superoxide dismutase (manufactured by Sigma, 23500
7■) 20■ were added and stirred at room temperature of 25°C for 2 hours.

Add 120 µl of acetic acid and 1 ml of monoethanolamine to this reaction solution.
After adding 20 μl and stirring for 20 minutes, the resulting solution was purified by ultrafiltration, condensed, and lyophilized to obtain modified superoxide dismutase.

The activity retention rate of this modified enzyme was 70%.

(2) Preparation of skin milk Using the above-mentioned modified SOD, an oil phase and an aqueous phase were prepared with the following raw material composition. Then, an aqueous phase also heated to 80°C was added to the oil phase heated to 80°C, and the mixture was rapidly cooled while stirring uniformly to obtain skin milk.

Oil phase liquid paraffin 20.00% cetyl alcohol 5. OO〃Polyoxyethylene sorbitan monooleate 5. OO〃
Water phase modified SOD 0.01% diglycerin 5. OO〃Methylparaben
0.10 Purified water 64.89
[Example 6] ■ Preparation of modified SOD Pullulan (average molecular weight 50,000
) was used. Modified superoxide dismutase was obtained in the same manner as in Example 1 except for the above.

The activity retention rate of this modified enzyme was 82%.

(2) Preparation of skin cream Using the above-mentioned modified SOD, an oil phase and an aqueous phase were prepared with the following raw material composition. Then, an aqueous phase also heated to 80°C was added to the oil phase heated to 80°C, and the mixture was quickly cooled while stirring uniformly to obtain a skin cream.

Oil phase Octyldodecyl myristate 35,00% Cetyl alcohol 5.00 Cetyl palmitate 2.00 Sorbitan sesquistearate 3, OO Water phase modified SOD 0.01% Glycerin
l 5. OO〃Polyoxyethylene sorbitan monooleate (20E, O,) 5
．． OO.

Methylparaben 0.10 Purified water
34.89 [Comparative Example 1] Copper obtained from viable blood cells instead of modified SOD.

Zinc-type superoxide dismutase (manufactured by Sigma, 2
A skin lotion was obtained by using the raw material (350 U/■) as it was and uniformly mixing the raw materials as shown below.

SOD O, 003% Glycerin 10.000 Purified water 89.997 [Comparative Example 2] Using the same modified SOD as in Example 1, the following raw material composition was prepared and these were uniformly mixed. Got skin lotion.

Modified SOD O, 01% Purified Water 99.99 The six types of actual height measurements and two types of comparative height measurements obtained as described above were evaluated by conducting each test according to the procedure described above. The results are shown in the table below.

(Left below) As can be seen from this table, all of the measurements performed have shown excellent effects in improving the skin. Then, skin J like Comparative Example 1 using unmodified superoxide dismutase
There is no irritation or rash on f.

In addition, the oral stability, which was a problem with the two products of Comparative Example, was not a problem at all in the actual measurement.

〔Effect of the invention〕

As described above, since the skin cosmetic of the present invention contains superoxide dismutase modified with polysaccharide and water-soluble polyhydric alcohol, it can be used to dry the skin without causing rough skin. , can prevent aging. Furthermore, this cosmetic has the advantage that its color and odor do not change orally and can be used safely for a long period of time.

Claims (5)

[Claims] (1) A skin cosmetic comprising as essential ingredients a modified superoxide dismutase in which a polysaccharide is bonded to a copper-zinc type superoxide dismutase and a water-soluble polyhydric alcohol. (2) The content of modified superoxide dismutase is
The skin cosmetic according to claim 1, wherein the enzyme amount is set to 0.00001 to 1% by weight. (3) The polysaccharide is at least one polysaccharide selected from the group consisting of dextran, starch, pullulan, inulin, xanthan gum, chitin, mannan, cellulose, pectin, carrageenan, and alginic acid, or The skin cosmetic according to item 2. (4) The skin cosmetic according to claim 3, wherein the polysaccharide is dextran with a molecular weight of 1,000 to 100,000. (5) Content of water-soluble polyhydric alcohol is 0.5 to 50
The skin cosmetic according to any one of claims 1 to 4, wherein the content is set to % by weight.